Calcium 3-hydroxy-4-[(4-methyl-2-sulphonatophenyl)azo]-2-naphthoate

Endpoint summary

Description of key information

Short term toxicity to fish:

Aim of this study was to investigate the effects of test chemical calcium 3-hydroxy-4-[(4-methyl-2-sulfonatophenyl) diazenyl]-2-naphthoate (D & C Red no. 7) on the growth of fish. Study was conducted according to the OECD guideline 203. 0.5 mg/l DMSO were used as a solvent. Five different concentration were prepared which ranges from17.1-180 mg/l. Test performed in open system by following flow-through method. A group of 10 fishes were exposed to 5 nominal concentrations. After the exposure of test chemical for 96 hrs, the LC50 was determine to be 33 mg/l for Oryzias latipes. Based on the LC50, chemical calcium 3-hydroxy-4-[(4-methyl-2-sulfonatophenyl) diazenyl]-2-naphthoate was consider as toxic and classified as aquatic chronic 3 as per the CLP classification criteria.

Short term toxicity to aquatic invertebrates:

Detection of the effect of calcium 3-hydroxy-4-[(4-methyl-2-sulfonatophenyl) diazenyl]-2-naphthoate on the growth of daphnia magna by following standard OECD guideline. The test was performed in static system by following the OECD guideline. Daphnia magna exposed with the chemical for 24hrs. Based on the immobilization of daphnia magna by the chemical calcium 3-hydroxy-4-[(4-methyl-2-sulfonatophenyl) diazenyl]-2-naphthoate (D & C Red no. 7) for 24hrs, the EC50 was 280 mg/l. Based on the EC50, chemical was consider as nontoxic and not classified as per the CLP classification criteria.

Long term toxicity to aquatic invertebrates:

The Key study from secondary sources 2017 was conducted to investigate the effects of test chemical on the growth of aquatic invertebrates by providing the exposure period of 21 days. Study was conducted according to the OECD Test Guideline 202 in a semi-static system. Solvent DMSO and HCO-40 in the ratio of 9:1 were used.5 nominal concentrations ranges from 3-300 mg/l prepared and 4 replicates were used in which 10 organisms per replicate added. After the exposure of 21 days effect on the mortality and reproduction of daphnia magna were recorded. Based on the mortality rate of daphnia magna by the test chemical for 21 days, the LC50 was determine to be 9.7 mg/l and on the basis of reproductive rate EC50 was determine to be 9.1 mg/l. Based on the long term toxicity effect, the chemical was consider as nontoxic and not classified as per the CLP classification criteria.

Toxicity to aquatic algae and cyanobacteria:

Aim of this study was to evaluate the nature of test chemical calcium 3-hydroxy-4-[(4-methyl-2-sulfonatophenyl) diazenyl]-2-naphthoate, when comes in contact with the test organism Desmodesmus subspicatus (previous name: Scenedesmus subspicatus). Test was conducted according to the OECD guideline 201. The solution 1 g/l was prepared by dissolving red powder in DMSO. Test solutions 5mg/l were prepared by mixing the stock solution of the test sample with OECD growth medium. Effects on the growth rate of the organism were studied at 5 mg/l concentration. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. The differences in means of control and sample were estimated by the t-test for independent groups at a 95 % confidence level, all individual replicates were used (STATISTICA CZ – data analysis software system, version 9.0, StatSoft, Inc.). Statistically significant differences are for p < 0.05. Effect on the growth of algae was determine after an exposure period of 72 hrs. Based on the growth rate inhibition of algae Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) due to the exposure of chemical calcium 3-hydroxy-4-[(4-methyl-2-sulfonatophenyl)diazenyl]-2-naphthoate only 3% inhibition were observed at 5 mg/l. on the basis of 3 % inhibition of algal population chemical might not be consider as toxic and classified thus chemical calcium 3-hydroxy-4-[(4-methyl-2-sulfonatophenyl) diazenyl]-2-naphthoate was consider as may me nontoxic and not classified as per the CLP criteria.

 

Toxicity to microorganisms:

1. The toxicity of test chemical was determined in terms of EC20 (% dilution) was 44.6 ± 11.6.. According to the ranking scheme for Microtox assay using EC20 values, test chemical can be categorized under moderately toxic category.

2. The death rate of the test organism at 10000 mg/l was 77.4%. Therefore the Effective concentration causing more than 50% death of Paramecium caudatum is reported as 10000 mg/l

Thus based on both the studies chemical calcium 3-hydroxy-4-[(4-methyl-2-sulfonatophenyl)diazenyl]-2-naphthoate (CAS no. 5281-04-9) was consider as nontoxic.

Additional information

Summarized result of toxicity of calcium 3-hydroxy-4-[(4-methyl-2-sulfonatophenyl) diazenyl]-2-naphthoate (CAS no. 5281-04-9) on the growth and other activity of fish, aquatic invertebrates, algae and cyanobacteria and microorganisms by considering the data for target as well as read across chemicals which was selected on the basis of structure and functional similarity are as follows:

 

Short term toxicity to fish:

Based on the experimental data for the target compound calcium 3-hydroxy-4-[(4-methyl-2-sulfonatophenyl) diazenyl]-2-naphthoate (CAS no. 5281-04-9) and experimental studies for supporting studies for its structural and functional similarity were reviewed for the short term toxicity on the fish end point which are summarized as below: 

 

In the first key study for the target chemical calcium 3-hydroxy-4-[(4-methyl-2-sulfonatophenyl) diazenyl]-2-naphthoate from secondary sources 2017 toxicity was measured on fish. Aim of this study was to investigate the effects of test chemical calcium 3-hydroxy-4-[(4-methyl-2-sulfonatophenyl) diazenyl]-2-naphthoate (D & C Red no. 7) on the growth of fish. Study was conducted according to the OECD guideline 203. 0.5 mg/l DMSO were used as a solvent. Five different concentration were prepared which ranges from17.1-180 mg/l. Test performed in open system by following flow-through method. A group of 10 fishes were exposed to 5 nominal concentrations. After the exposure of test chemical for 96 hrs, the LC50 was determine to be 33 mg/l for Oryzias latipes. Based on the LC50, chemical calcium 3-hydroxy-4-[(4-methyl-2-sulfonatophenyl) diazenyl]-2-naphthoate was consider as toxic and classified as aquatic chronic 3 as per the CLP classification criteria.

 

Similarly in the second study from the secondary sources supports the classification for fish. Aim of this study was to investigate the effects of test chemical calcium 3-hydroxy-4-[(4-methyl-2-sulfonatophenyl) diazenyl]-2-naphthoate (D & C Red no. 7) on the growth of fish. Study was conducted according to the Japanese Industrial Standard (JIS K 0 102- 1986-71). 0.5 mg/l DMSO were used as a solvent. Five different concentration were prepared which ranges from17.1-180 mg/l. Test performed in open system by following flow-through method. A group of 10 fishes were exposed to 5 nominal concentrations. Based on the mortality of fish Oryzias latipes by the chemical exposure calcium 3-hydroxy-4-[(4-methyl-2-sulfonatophenyl) diazenyl]-2-naphthoate (D & C Red no. 7) for 48 hrs, the LC50 was determine to be 50 mg/l. Based on the LC50, chemical was consider as toxic and classified as aquatic chronic 3 as per the CLP classification criteria.

 

Above studies was supported by the third experimental study for the structural and functional similarity read across chemical (from experimental study report 2017). The study was designed to assess the toxic effects of the test compound on the Zebra Fish (Danio rerio). Study was conducted according to the OECD Guideline 203 (Fish, Acute Toxicity Test). The test substance was soluble in water. Therefore, the test solution was prepared by dissolving 1 g of the test substance in 1 liters of potable water (passed through reverse osmosis system) with 2 hours continuous stirring for achieving test concentrations of 6.25mg/L, 12.5mg/L, 25mg/L, 50mg/L,100mg/L ,respectively. The test vessels used were all plastic aquaria having the water capacity of 5liters. Bowl aquaria containing 2 liters of potable water (passed through reverse osmosis system) were loaded with 8 fishes each. A static procedure was used for the study. The experimental phase of the main study was for a period of 96 hrs. Observations (mortality, visible symptoms, pH, Temperature, dissolved oxygen content) were recorded after 24, 48, 72 and 96 hours of the start of the experiment. The fishes were moving slowly as compared to control. No mortalities were observed in the control aquaria. The lethal concentrations LC50 was determine to be 50 mg/L. As the experiment was conducted according to the OECD guideline and fulfill all the necessary criteria required for standard, thus it was consider as valid.

LC0 (96 hours) (highest loading at which no mortality was observed) = 25 mg/L

LC50 (96 hours) Experimental = 50 mg/L

LC100 (96 hours) (lowest loading at which 100% mortality was observed) = 100 mg/L

This value indicates that the substance is toxic to fish and can be classified as Aquatic chronic category 3.

 

Thus based on the above results for the chemical from various sources, it can be concluded that the test chemical calcium 3-hydroxy-4-[(4-methyl-2-sulfonatophenyl) diazenyl]-2- naphthoate (CAS no. 5281-04-9) was consider as toxic and classified as aquatic chronic 3 as per the CLP classification criteria.

 

Short term toxicity to aquatic invertebrates:

Based on the experimental data for the target compound calcium 3-hydroxy-4-[(4-methyl-2-sulfonatophenyl) diazenyl]-2-naphthoate (CAS no. 5281-04-9) and experimental studies for supporting studies for its structural and functional similarity were reviewed for the short term toxicity on the aquatic invertebrates end point which are summarized as below: 

 

In the first key study for the target chemical calcium 3-hydroxy-4-[(4-methyl-2-sulfonatophenyl) diazenyl]-2-naphthoate from secondary sources toxicity was measured. Detection of the effect of calcium 3-hydroxy-4-[(4-methyl-2-sulfonatophenyl) diazenyl]-2-naphthoate on the growth of daphnia magna by following standard OECD guideline. The test was performed in static system by following the OECD guideline. Daphnia magna exposed with the chemical for 24hrs. Based on the immobilization of daphnia magna by the chemical calcium 3-hydroxy-4-[(4-methyl-2-sulfonatophenyl) diazenyl]-2-naphthoate (D & C Red no. 7) for 24hrs, the EC50 was 280 mg/l. Based on the EC50, chemical was consider as nontoxic and not classified as per the CLP classification criteria.

 

First study was supported by the second experimental study for the structural and functional similarity read across chemical (from experimental study 2016). Determination of the inhibition of the mobility of daphnids was carried out with the test substance according to OECD Guideline 202. A limit test at sample concentration of 100 mg/L was performed. Effects on immobilisation were observed for 48 hours. The median effective concentration (EC50) for the test substance, in Daphnia magna was determined to be >100 mg/L for immobilisation effects. Based on this EC50 value and after comparing with CLP criteria for aquatic classification of the substance it is concluded that the substance, does not exhibit short term toxicity to aquatic invertebrate (Daphnia Magna) and not classified.

 

Similarly in the third study for the structurally similar read across chemical toxicity was measured (from experimental study 2016). Determination of the inhibition of the mobility of daphnids was carried out with the substance according to OECD Guideline 202. The limit test was performed at 100 mg/l. Effects on immobilisation were observed for 48 hours. The solution 100 mg/l was prepared by dissolving brown powder in reconstituted water. The effective concentration (EC8) for the test substance, in Daphnia magna was determined to be 100 mg/L on the basis of mobility inhibition effects in a 48 hour study. This value indicates that the substance is likely to be non-hazardous to aquatic invertebrates and cannot be classified as toxic as per the CLP criteria.

 

Based on the above experimental data from various sources, chemical calcium 3-hydroxy-4-[(4-methyl-2-sulfonatophenyl) diazenyl]-2-naphthoate (CAS no. 5281-04-9) was consider as nontoxic and not classified as per the CLP classification criteria.

 

Long term toxicity to aquatic invertebrates:

The Key study from secondary sources 2017 was conducted to investigate the effects of test chemical on the growth of aquatic invertebrates by providing the exposure period of 21 days. Study was conducted according to the OECD Test Guideline 202 in a semi-static system. Solvent DMSO and HCO-40 in the ratio of 9:1 were used.5 nominal concentrations ranges from 3-300 mg/l prepared and 4 replicates were used in which 10 organisms per replicate added. After the exposure of 21 days effect on the mortality and reproduction of daphnia magna were recorded. Based on the mortality rate of daphnia magna by the test chemical for 21 days, the LC50 was determine to be 9.7 mg/l and on the basis of reproductive rate EC50 was determine to be 9.1 mg/l. Based on the long term toxicity effect, the chemical was consider as nontoxic and not classified as per the CLP classification criteria.

 

Toxicity to aquatic algae and cyanobacteria:

 

Based on the experimental data for the target compound calcium 3-hydroxy-4-[(4-methyl-2-sulfonatophenyl) diazenyl]-2-naphthoate (CAS no. 5281-04-9) studies were reviewed for the toxicity on the algae end point which are summarized as below: 

 

In the first study from experimental lab report 2018, study was to evaluate the nature of test chemical calcium 3-hydroxy-4-[(4-methyl-2-sulfonatophenyl) diazenyl]-2-naphthoate, when comes in contact with the test organism Desmodesmus subspicatus (previous name: Scenedesmus subspicatus). Test was conducted according to the OECD guideline 201. The solution 1 g/l was prepared by dissolving red powder in DMSO. Test solutions 5mg/l were prepared by mixing the stock solution of the test sample with OECD growth medium. Effects on the growth rate of the organism were studied at 5 mg/l concentration. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. The differences in means of control and sample were estimated by the t-test for independent groups at a 95 % confidence level, all individual replicates were used (STATISTICA CZ – data analysis software system, version 9.0, StatSoft, Inc.). Statistically significant differences are for p < 0.05. Effect on the growth of algae was determine after an exposure period of 72 hrs. Based on the growth rate inhibition of algae Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) due to the exposure of chemical calcium 3-hydroxy-4-[(4-methyl-2-sulfonatophenyl)diazenyl]-2-naphthoate only 3% inhibition were observed at 5 mg/l. on the basis of 3 % inhibition of algal population chemical might not be consider as toxic and classified thus chemical calcium 3-hydroxy-4-[(4-methyl-2-sulfonatophenyl) diazenyl]-2-naphthoate was consider as may me nontoxic and not classified as per the CLP criteria.

 

 

Similarly in the second study from secondary sources detection of the effect of test chemical on the growth of Selenastrum capricornutum ATCC 22662 by following standard 202 OECD guideline. The test was performed in static system by following the OECD guideline. Selenastrum capricornutum ATCC 22662 exposed with the chemical for 72hrs. Based on the biomass inhibition of Selenastrum capricornutum ATCC 22662 by the chemical calcium 3-hydroxy-4-[(4-methyl-2-sulfonatophenyl) diazenyl]-2 -naphthoate for 72hrs, the NOEC was 5.8 mg/l, and the EC50 was 190 mg/l. Based on the EC50 value it was concluded that the chemical calcium 3-hydroxy-4-[(4-methyl-2-sulfonatophenyl) diazenyl]-2-naphthoate was nontoxic and can be consider to be not classified as toxic as per the CLP classification criteria.

 

Based on the above experimental data chemical calcium 3-hydroxy-4-[(4-methyl-2-sulfonatophenyl) diazenyl]-2-naphthoate (CAS no. 5281-04-9) was consider as nontoxic and not classified as per the CLP classification criteria.

Toxicity to microorganisms:

Toxicity of calcium 3-hydroxy-4-[(4-methyl-2-sulfonatophenyl)diazenyl]-2-naphthoate (CAS no. 5281-04-9) was studied on the growth and other biological activity of microorganisms is predicted on the basis of it structurally and functionally similar read across chemicals. The studies are as mentioned below:

 

Toxicity study was performed to determine the toxic effect of the structurally and functionally similar read across chemicals on the microorganisms (From peer reviewed journal, 2002). The Microtox acute toxicity assay was performed by using a modified strain of Vibrio fischeri. Frozen samples were brought to room temperature, and centrifuged. The pH of the samples was adjusted where necessary to 6 by adding 0.5 ml 0.58 M KH2 PO4 and 70μl 1 M NaOH colour correction was done at 490 nm. The Microtox acute toxicity assay was performed in a Microtox 500 Analyzer on samples before and after decoloration according to the test protocols defined by the manufacturer from eight serial dilutions, the percent concentration to decrease 20% of the luminescence of a modified strain of Vibrio fischeri (EC20) after 5 min incubation was calculated with the Microtox data analysis program. A solution of 1 g/l ZnSO4·7H2O was used as the positive control and 1 g/l glucose as the negative control. Each EC20 reported is the average of triplicate analysis.

The concentration to decrease 50% of the bacterial luminescence in the Microtox acute assay (EC50) is normally reported. However, in most of these studies, the EC50 before or after decoloration was greater than 100% indicating that there was no toxicity or toxicity change. To better evaluate whether the decoloration process affected toxicity, the dilution required to decrease 20% of the bacterial luminescence relative to the control (EC20) was reported instead. The following rating was adapted. –

EC20: >100%=nontoxic;

>75–100%=slightly non-toxic;

 >50–75%=toxic;

>25–50%=moderately toxic;

<25% very toxic.

The toxicity of 100mg/l of test chemical determined in terms of EC20 (% dilution) was 44.6 ± 11.6.

 

Similar study was performed for another structurally and functionally similar read across chemicals (from peer reviewed journal 1977). The death of Paramecium caudatum (PC), a unicellular animal, can be observed more readily and in far less time than that of small animals. Hence a bioassay was conducted to study the toxic effect of test chemical. Paramecium Caudatum was maintained at 22°C on 0.15 % dried lettuce infusion and fed with Aerobacter aerogenes. Chemical was tested in 0.1% and 1% concentration. The test concentrations were put in a hollow slide glass, and an equal volume of 0.04 M phosphate buffer, pH 7.0, was added. After 5 to 10 test organisms were added, their survival times were measured microscopically. Thirty to forty test organisms for each concentration were tested by the same method, and the mean survival time and the death rate were calculated. The survival time was defined as the time required until death was observed for each concentration. Death was assumed to have occurred when there was no movement. The death rate was defined as the percentage of deaths observed during 20 minutes. The mean survival time (in sec) of test organism Paramecium caudatum was determined to be 695 seconds.  The death rate of the test organism at 10000mg/l was 77.4%. Therefore the Effective concentration causing more than 50% death of Paramecium caudatum was reported as 10000 mg/l.

Thus based on both the studies chemical calcium 3-hydroxy-4-[(4-methyl-2-sulfonatophenyl)diazenyl]-2-naphthoate (CAS no. 5281-04-9) was consider as nontoxic.

Based on the above toxicity data of test chemical, it was concluded that the 5281 -04 -9 was nontoxic to invertebrates, algae and microorganisms but it was toxic to fish. Thus based on the effects on the growth of fishes chemical was consider as toxic and classified as aquatic chronic 3 as per the CLP classification criteria.